A TiO₂ modified abiotic–biotic process for the degradation of the azo dye methyl orange

Abstract

To investigate the feasibility of titanium dioxide (TiO₂) employed as a modifier in the sodium alginate immobilization system, the degradation of methyl orange with the strain Delftia sp. A2(2011) (STT01) was carried out using a TiO₂ modified sodium alginate system (TiO₂/SA) and a non-TiO₂ modified sodium alginate system (SA). It was found that the decolorization of methyl orange was enhanced from 76.5% to 100%, and the chemical oxygen demand (COD) removal was increased from 35.6% to 52.7%. The results further revealed that the TiO₂ played a crucial role in the cell immobilization system, and the potential modification mechanisms of dye sensitization and TiO₂–SA complex-mediated photocatalysis were investigated. Additionally, the intrinsic bright color of the bacterial strain STT01 could be ingeniously employed as an indicator for the degradation efficiency. This work not only presents a promising opportunity for developing novel cell immobilization techniques but also affords a direct and visually observed treatment for azo dye wastewater.

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1. Introduction

It has been reported that approximately 10 000 different dyes and pigments are industrially used and over 0.7 million tons of synthetic dyes are annually produced on Earth.^1,2 Among them, azo dyes are ca. one half of these dyes practically employed in the textile industry.^3–5 Therefore, decolorization of azo dye effluents has received considerable attention in the past few decades due to their stability, toxicity and resistance to aquatic and terrestrial organisms.^6–8

Traditional physical or chemical techniques such as adsorption and chemical precipitation are unfavorable for dye treatment because these methods can generate a secondary pollution originating from organic compound transfer.^9–11 Although advanced oxidation processes (AOPs) can oxidize a broad range of pollutants, they cannot be employed to effectively degrade azo dyes due to their relatively high cost.^12–15

Microbial or enzymatic decolorization is known to be an eco-friendly and cost-competitive alternative to the chemical decomposition processes, but the toxic and genotoxic effects of azo dyes to microorganisms restrict the use of common biological treatments.^2,16,17 Therefore, developing novel strategies for the degradation of azo dyes is believed to be essential.

As is well known, cell immobilization is a feasible technique for continuous degradation due to the high treatment efficiency, preferential retention of biomass, convenience of bacteria–substrate separation and avoidance of product inhibition, compared with those of the free cells.^18–20 In general, immobilization supports consist of inorganic materials and/or organic materials, such as ceramics,^21,22 agar, agarose, k-carrageenan or sodium alginate gel,^23,24 and fibers.²⁵ Among all of the matrices, alginate extracted from macro-algae, a natural polymer, is widely used for the preparation of gel beads possessing low cost, higher bioactivity and mild conditions of immobilization.

The microorganisms immobilized in alginate beads have been used in hostile environments and the beads provide nutrients and appropriate conditions, such as facilitating the transfer of oxygen which is crucial for rapid hydrocarbon mineralization, to allow for rapid bioremediation in contaminated systems.^26–30 However, the dense gel layers of the sodium alginate beads can hinder the mass transfer of substrates, pollutants, and degraded products. Furthermore, the resulting beads in the sodium alginate-immobilized system are relatively weak in mechanical strength and stability,^24,31 limiting their practical application. So, the incorporation of some modifiers into the alginate beads could facilitate the transport of pollutants towards both the surface and interior regions.³²

TiO₂, as an effective photosensitizer, has traditionally performed as a catalyst under irradiation in abiotic processes for pollutant degradation, generating strong oxidants such as HO˙ radicals that can quickly and non-selectively degrade organic compounds.^33–36 The promising applications of TiO₂ have been investigated in numerous fields ranging from photovoltaics and photocatalysis to photo-/electrochromics and sensors. In most of the reported cases the performance depended not only on the properties of the TiO₂ material itself but also on the modifications of the TiO₂ material host, and on the interactions between TiO₂ and the environment.^37–41 Whereas, the application of TiO₂ as a microorganism immobilization support has been little documented and its application for biotic/abiotic degradation has not been yet described.

Therefore, the aim of this work is to explore a novel biotic/abiotic process initiated by TiO₂/SA, in which TiO₂ plays a great role in modifying the performance of the SA immobilized system. The focus was on evaluating its feasibility for the decolorization and degradation of the azo dye methyl orange. The degradation mechanisms of methyl orange were probed via scanning electron microscopy (SEM), optical images, UV-vis, IR and LC-MS analyses, and the promotion mechanisms involved in the TiO₂/SA process were further elucidated.

2. Materials and methods

2.1 Materials

Methyl orange (C₁₄H₁₄N₃NaO₃S), H₂SO₄, NaOH, 2-propanol, anatase titanium dioxide (TiO₂, with a surface area of ca. 6.6 m² g⁻¹), SA((C₆H₇NaO₆)_n with a mean molecular weight of 500 kDa, a particle size less than 200-mesh, and the ratio of mannuronate residues to guluronate residues (M/G) of 0.50), ammonium acetate, and the reagents used for the bacterial medium, were all purchased from Sinopharm Chemical Reagent Co., Ltd., China, and were of analytical grade; methanol and acetonitrile were of chromatographic purity grade. All of the reagents were used as received.

The applied basal medium contained the following: per liter of ultra-pure water, 2.5 g CH₃COONa, 0.6 g KH₂PO₄, 0.9 g K₂HPO₄, 0.075 g CaCl₂·2H₂O, 0.20 g yeast extract, 0.40 g MgCl₂, 0.36 g NH₄Cl, 0.24 mg ZnSO₄·7H₂O, 2.8 mg H₃BO₃, 0.75 mg Na₂Mo₄·2H₂O, 11.8 mg FeSO₄·7H₂O, and 0.04 mg Cu (NO₃)₂·3H₂O. After adjusting the pH to 7.0 with 1.0 mol L⁻¹ NaOH, the medium and the vessel were sterilized at 121 °C for 30 min. Ultra-pure water was provided by a Milli-Q system (Millipore).

2.2 Microorganism

The strain STT01 used in the work was isolated from the activated sludge of the urban sewage treatment plant in Changsha, China, according to the following method: sludge samples were agitated to obtain homogeneous suspensions in 0.9% sterile NaCl. 1 mL suspensions were pipetted into a 10 mL screw-cap tube. The tube was then completely filled with the basal medium and tightly closed in order to keep them under microaerophilic conditions with dissolved oxygen (DO) of 0–0.5 mg L⁻¹. The incubation was performed at 30 °C under an illumination intensity of 3000 lux until the culture medium turned bright red, which indicated that a large amount of bacteria cells had been produced. The bacteria cells harvested from the reactor were further cultivated in a batch culture fed with basal medium containing 500 mg L⁻¹ methyl orange as the carbon source. The strain was isolated on solid media consisting of basal medium, 1.5% agar and 500 mg L⁻¹ methyl orange, which were added as a sterile solution to the autoclaved media. Colonies exhibiting strong growth were re-streaked an additional six times and then placed into basal medium containing methyl orange for storage. Transfers into fresh medium were carried out at weekly intervals. Observation under a microscope indicated that the strain STT01 formed a red colony on facultative anaerobic medium, and the cells of the strain were Gram-negative, and short-rod with flagellum. The optimum conditions for cell growth and biodegradation were investigated and found to be pH 7.0; illumination intensity, 3000 lux (the light source used in the work was an incandescent lamp, which had mixed wavelengths in the range of 400–780 nm); temperature, 30 °C; DO, 0–0.5 mg L⁻¹. The complete sequence of the 1454 bp 16S rDNA fragment form of STT01 has been deposited in the Gene Bank database under accession number HQ659695.

2.3 Cell immobilization

The immobilization process was conducted according to the immobilization procedure presented by Nagadomi et al.^21,22 with some revisions.

The wet cells for immobilization were harvested at the end of the exponential growth phase. The weight of the initial wet mass of the cells was fixed at 2.0 g, which were harvested by centrifugation of 400 mL cell suspension (OD₅₉₀ = 1.5) for 15 min with a speed of 5000 rpm. The TiO₂/SA support was prepared as follows: 35.0 g L⁻¹ aqueous solution of SA was first prepared by stirring 8.75 g SA in 250 mL ultra-pure water for 24 h. The required amount of TiO₂ was taken in 10 mL water and sonicated for 2 h, and then the TiO₂ dispersion was added to the SA aqueous solution and stirred overnight. The mixture was further sonicated to drive off bubbles, autoclaved for 2 min, and cooled down to room temperature.

The immobilized cell beads were prepared by extrusion spheronization: the harvested cells were taken with the prepared support TiO₂/SA and stirred carefully to remain under homogenous conditions, and the mixture was directly dropped into 4% (w/v) CaCl₂ with a 50 mL disposable syringe at 10 cm height from the liquid level. The resulting beads with diameters of 3.0–4.0 mm were then held for 24 h at 4 °C to harden the immobilization. To achieve the optimal immobilization conditions, the TiO₂ quantity (1.0, 1.5, 2.0, 2.5, and 3.0 g L⁻¹), and the mass ratio of wet cells to immobilization support (C : IS, 1 : 5, 1 : 10, 1 : 20, 1 : 30, and 1 : 40, w/v) were investigated. The immobilized beads were washed with sterile water twice and divided into nine aliquots for the degradation experiments. The control experiments with a SA immobilized system were conducted under the same conditions used for TiO₂/SA.

2.4 Degradation experiments

The degradation of methyl orange using STT01 was carried out with the following systems: TiO₂/SA, SA, free cells and free cells suspended with TiO₂. Continuous degradation experiments of methyl orange were carried out in order to investigate the continuous biodegradation of the immobilized beads. At the end of each run cycle, the degraded effluent was discharged, the immobilized beads were washed twice with sterile water, and the weight of wet biomass was determined through weighing the immobilized cell beads after being dried using sterile filter paper in a clean bench (SWCJ1FD, Antai, Jiangsu) before and after the respective run cycle. All degradation experiments were carried out in triplicate in 250 mL tapered conical flasks that contained 10 mL culture medium. In the free cell system, the same amount of wet free cells was used instead of immobilized beads for the degradation. Methyl orange (1000 mg L⁻¹) was degraded for 96 h; during the degradation process, the decolorization and COD_Cr treatment efficiency were measured using the potassium dichromate (K₂Cr₂O₇) method⁴⁴ at intervals of 12 h.

To investigate the degradation mechanisms, control experiments in the absence of STT01 and of TiO₂ in the dark; TiO₂ under irradiation; SA beads in the dark; SA beads under irradiation; TiO₂/SA beads in the dark and TiO₂/SA beads under irradiation were further investigated.

2.5 HO˙ scavenging and verification studies

The investigation of HO˙ scavenging and verification was carried out according to the literature.^42,43 2-Propanol was used as the scavenger. The molar ratio of scavenger to TiO₂ was 50 : 1. The degree of methyl orange degradation was verified by evaluating the decolorization and COD removal efficiency.

2.6 Analysis

The samples used for COD analysis were collected after centrifuging at 10 000 rpm for 20 min to remove the free cells. The COD measurements were conducted according to standard methods.⁴⁴ The pH was adjusted to 7.0 using 1.0 mol L⁻¹ H₂SO₄ or 1.0 mol L⁻¹ NaOH and measured with a pH meter (pH330i, WTW Germany). The DO was controlled with a N₂ flow and measured with a DO detector (Oxi300i, WTW Germany). The samples for SEM analysis were prepared via freeze-drying the corresponding harvested cells and immobilized support (TiO₂, SA) under vacuum at −45 °C. The degraded effluent for UV-vis and IR spectroscopy was first dried via freeze-drying under vacuum at −45 °C, and the resulting solids were then dissolved in methanol to remove inorganic matter and filtered using a Millipore filter (pore size of 0.22 μm), and dried again via freeze-drying under vacuum at −45 °C. IR spectra were recorded on a Perkin-Elmer FTIR spectrophotometer (USA) using KBr pellets. The samples for UV-vis spectroscopy and LC-MS were further dissolved in ultrapure water. UV-vis spectra were recorded using a UV-vis spectrophotometer (UV2550, Japan) with a 1 cm quartz cell.

The samples for LC-MS detection were taken from the fourth run cycle with the maximum degradation efficiency in the TiO₂/SA immobilized system. LC-MS experiments were conducted using a Thermo Finnigan LCQ-Advantage (USA) referring to the method presented by Baiocchi et al.³ HPLC analyses were carried out under isocratic conditions using a RP-C18 column (Lichrospher RP-18, 250 mm × 4.6 mm; 5 mm particles, Merck, Darmstadt, Germany) and the mobile phase was composed of 10 mM acetonitrile–ammonium acetate at pH 6.8 (20/80 (v/v)), and the flow rate was 0.8 mL min⁻¹. The eluent from the chromatographic column could successively enter the UV-vis diode array detector, the electrospray ionization (ESI) interface and the dual ion trap mass analyzer. The ESI was in the negative mode with dual sprayers of the ion trap MS source, operated under the conditions of a drying gas at 6.0 L min⁻¹, gas temperature of 300 °C, nebulizer of 1 kPa, ion current control of 150 000, maximum accumulation time of 50 ms, scan of 100–325, cone gas flow of 55 L h⁻¹, desolvation temperature of 300 °C, ion source temperature of 100 °C, desolvation gas flow of 400 L h⁻¹, and capillary of 3800 V.

A SEM equipped with an energy dispersive spectrometer (EDS) (Quanta 200, FEI, Germany) was used to characterize the immobilized beads for their basic constituents and morphological information.

2.7 Statistical analysis

To ensure the accuracy of the test data, each experiment was performed in triplicate within ±10% data deviation to ensure reproducibility. The average values were used in the analysis.

3. Results and discussion

3.1 Optimum conditions investigations for the degradation of methyl orange

Fig. 1 shows the biodegradation kinetics of methyl orange by free STT01 at different initial concentrations. The results showed that methyl orange could be completely decolorized with the initial concentrations of 100 mg L⁻¹ and 500 mg L⁻¹, while the degree of decolorization apparently decreased when the initial concentration was increased to 1000 mg L⁻¹. Therefore, in contrast to the free STT01, 1000 mg L⁻¹ of methyl orange was chosen as the basal concentration in the cell immobilization system.

Fig. 1 Kinetics of methyl orange biodegradation by the free strain STT01.

To achieve the optimal immobilization conditions, the quantity of TiO₂ and the mass ratio of wet cells to immobilization support (C : IS, w/v) were investigated. In Fig. 2a, with the increasing quantity of TiO₂, the treatment efficiency gradually increased until the quantity of TiO₂ was enhanced to 2.0 g L⁻¹. Conversely, the treatment efficiency decreased when the quantity of loaded TiO₂ was more than 2.0 g L⁻¹. This could be ascribed to the dense gel layers of the TiO₂/SA beads that could hinder the microorganism accumulation and mass/energy transfer of the substrates, pollutants, and degraded products, inhibiting the cell growth and bioactivity.^30–32,45 On the other hand, the excess quantity of TiO₂ had bactericidal effects on the cells, reducing the active biomass.^39,41 So, the optimal quantity of TiO₂ was selected as 2.0 g L⁻¹.

Fig. 2 The treatment efficiency with varying quantities of TiO₂ in the TiO₂/SA system (a); ratios (w/v) of C : IS in the TiO₂/SA and SA system (b). Note that C : IS is the ratio of the wet cells to immobilization support (C : IS, w/v); the quantity of TiO₂ is the mass of TiO₂ that is mixed with SA as the immobilization support in the TiO₂/SA system.

In Fig. 2b, the optimal treatment efficiencies were obtained at a ratio of C : IS of 1 : 20. As the immobilization support, TiO₂/SA or SA was responsible for the mass and energy transfer for the immobilized cells. The inadequate loadings of C : IS (such as 1 : 5 or 1 : 10 for the ratio of C : IS) could have had an inhibition effect on cell growth due to the ineffective mass/energy transfer of the cells inside the beads, and thus biodegradation was just carried out on the surface of the beads, decreasing the bioactivity and reducing the treatment efficiency.^30–32 On the contrary, when the ratio of C : IS was changed to 1 : 30 or 1 : 40, the excess loading of TiO₂/SA or SA would cause a eutrophic environment, which also had an inhibitory effect on cell growth. Especially, the excess loading of TiO₂/SA would bring an excess quantity of TiO₂, which had bactericidal effects on the cells, reducing the active biomass.⁴¹ So the optimal ratio of C : IS was selected as 1 : 20.

3.2 Various processes for the degradation of methyl orange

Under the optimal conditions, the maximum decolorization and COD removal reached 100% and 52.7% in the TiO₂/SA system respectively , whereas they reached 76.5% and 35.6% in the SA system; 54.5% and 20.9% in the free cell system; 33.2% and 6.9% in the free cell system with TiO₂ (Fig. 3a–d), respectively. As expected, the TiO₂/SA system exhibited much more excellent biodegradation of methyl orange.

Fig. 3 Treatment efficiency of methyl orange in the TiO₂/SA system (a), SA system (b), free cell system and free cell system suspended with TiO₂ under the optimal conditions.

The control experiments in the absence of STT01 were conducted simultaneously (Fig. 4). The results revealed that TiO₂, irradiation and SA played a great role in the degradation process. Compared with the results in Fig. 3a–d, it was found that STT01 was responsible for the large-scale treatment efficiency.

Fig. 4 The comparative methyl orange treatment efficiency in various processes in the absence of STT01 after 96 h of incubation. Processes: (1) TiO₂ in the dark; (2) TiO₂ under irradiation; (3) SA beads in the dark; (4) SA beads under irradiation; (5) TiO₂/SA beads in the dark; (6) TiO₂/SA beads under irradiation.

3.3 Degradation pathway of methyl orange in the TiO₂/SA system

The UV-vis spectrum of methyl orange shows two peaks located at 466 nm and 269 nm before treatment (Fig. 5). After 96 h of degradation, the absorption peak originating from the chromophore of the N=N group (466 nm) decreased quickly, while the absorption at 269 nm originating from the benzene ring simultaneously blue shifted to 245 nm, indicating that the azo bond was destroyed and other degradation intermediates containing the benzene ring were generated. It was evident that the decolorization of the methyl orange was easily achieved while the destruction of the benzene ring was hardly accomplished.

Fig. 5 UV-vis spectra of the original and degraded methyl orange in the TiO₂/SA immobilized system.

In Fig. 6, the original IR spectrum of methyl orange exhibited a peak at 3437 cm⁻¹ for the N–H stretching vibration; the C–H stretching vibrations of –CH₃ were located at 2924 cm⁻¹ and 2854 cm⁻¹; the C–C vibration of the benzene skeleton was observed at 1609 cm⁻¹ and 1542 cm⁻¹; the N=N vibration appeared at 1421 cm⁻¹; the C–N vibrations were assigned at 1369 cm⁻¹ and 1194 cm⁻¹; the S=O vibration was located at 1122 cm⁻¹; the C–H stretching vibrations of the benzene ring were located at 1039 cm⁻¹, 847 cm⁻¹, 818 cm⁻¹ and 698 cm⁻¹; the C–S stretching vibrations were observed at 624 cm⁻¹ and 575 cm⁻¹.

Fig. 6 IR spectra of the original methyl orange and the degraded products with TiO₂/SA immobilized STT01.

The degraded products of methyl orange show a peak at 3453 cm⁻¹ for the N–H bend, a band at 3130 cm⁻¹ due to the presence of aromatic C–H bonds, which were not present in the original methyl orange but appeared in the degraded product. This could be ascribed to the conjugated double bond of N=N in the original methyl orange weakening the absorbance. This further indicated that the conjugated π–π interactions resulted from the azo bond N=N which was broken down.³ A peak at 2926 cm⁻¹ for asymmetric –CH₃ stretching vibrations; peaks at 1578 cm⁻¹, 1462 cm⁻¹ for C–C vibration of the benzene skeleton; 1416 cm⁻¹ and 1261 cm⁻¹ for C–N aromatic stretching vibrations; 1122 cm⁻¹ and 1070 cm⁻¹ for S=O stretching vibrations; 956 cm⁻¹ and 849 cm⁻¹ for aromatic C–H vibration; 575 cm⁻¹ for C–S, indicated the formation of new products with a sulfonated aromatic ring or benzene ring.

The results were further supported by LC-MS (Fig. 7), the degraded products were separated at the retention times of 5.59 min and 8.52 min (Fig. 7a), and each peak was characterized using its mass measurements (m/z) (Fig. 7b–c). It was concluded that the degradation pathway of methyl orange firstly involved in a symmetric cleavage of the azo bond yielding benzenesulfonic acid at m/z 157 (C₆H₅SO₃) (Fig. 7b). The other degradation product was N,N′-dimethyl benzenamine, which was confirmed via a mass measurement at m/z 121 (C₈H₁₁N) with a characteristic fragment at m/z 106 (Fig. 7c). Therefore, the degradation pathway of methyl orange potentially involved a cleavage of the azo bond, yielding benzenesulfonic acid and N,N′-dimethyl benzenamine (Fig. 7d), which was then followed by further degradation or mineralization.

Fig. 7 LC chromatogram of the degraded methyl orange (a); the mass spectra and compound confirmation for the degradation products (b and c); degradation pathway of methyl orange (d).

3.4 Characteristics of the TiO₂/SA system

Fig. 8 demonstrates that the wt% of Ti in the sodium alginate was 0.46%, which increased to 16.25% in the immobilized beads after the fifth treatment. This indicated that the increasing content of Ti must come from the addition of TiO₂. As shown in Fig. 9a–c, the structures of TiO₂ and the SA particles are full of cavities that could provide preferential pathways for microorganism accumulation and mass transfer.^26,28,31,38 Fig. 9d presents that the immobilization support was filled with Zoogloea after the fifth run cycle. The results firmly demonstrated that the strain STT01 could not only survive in the TiO₂/SA system but also perform continuous biodegradation of methyl orange due to the appreciable removal efficiency in the five run cycles (Table 1), which could be further supported through the optical images of STT01 beads immobilized by TiO₂/SA (Fig. 10a–f). It was evident that the beads immobilized by TiO₂/SA had a brighter colour, possessed better granular characteristics and maintained continuous biodegradation. Whereas, some of the SA immobilized beads were softer and the cells were gradually released from the immobilized gels into the reaction liquid in the first degradation run cycle and the immobilized beads were difficult to use for carrying out the next run cycle (Fig. 10g–h). This result indicated that beads immobilized by SA were less stable than those immobilized by TiO₂/SA. It was strongly revealed that TiO₂ could improve the mechanical strength and stability of the beads.^38,40 Fig. 10i demonstrates that the cells of STT01 changed from bright red to black when TiO₂ was suspended in the free cell system, which declared that TiO₂ had a bactericidal effect on the free cells. The results are in agreement with those reported in the literature.^39,41,46

Fig. 8 Elemental analysis of the initial sodium alginate (a) and the beads in the TiO₂/SA system after the fifth treatment cycle (b).

Fig. 9 SEM images of the original SA (a); original TiO₂ (b); TiO₂/SA immobilized STT01 before treatment (c); TiO₂/SA immobilized STT01 after the fifth treatment cycle (d).

Table 1 Characteristics of the continuous run cycles

Treatment efficiency and biomass content^a	Run cycle
	Cycle 1	Cycle 2	Cycle 3	Cycle 4	Cycle 5
a The biomass content was described by the mass change of the beads after the corresponding run cycle. The initial biomass content was 28.5 g; sd, standard deviation.
CODcr removal (% ± sd)	41.2 ± 0.5	45.8 ± 0.7	52.5 ± 0.6	52.7 ± 0.5	51.9 ± 0.3
Biomass content (g ± sd)	30.4 ± 0.4	32.5 ± 0.3	35.8 ± 0.2	37.2 ± 0.4	37.0 ± 0.5

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Fig. 10 Optical images of the strain beads immobilized by TiO₂/SA: before treatment (a); after the 1st (b); 2nd (c); 3rd (d); 4th (e); and 5th (f) run cycle; optical images of the strain beads immobilized by SA: before treatment (g); after the first cycle (h); the color of the cells in the free cells system with/without TiO₂ (i). Note that the pictures of the beads (a) and (g) were taken in 4% CaCl₂ and the pictures (b)–(f) were taken in the corresponding degraded effluent. The pictures (g) and (h) were taken from the topside.

The biomass content could be qualitatively presented by the color change of the immobilized beads (Fig. 10a–f) and quantitatively depicted by the mass change of the immobilized beads before/after the corresponding run cycle (Table 1). The maximum mass of the immobilized beads in the TiO₂/SA system increased from ca. 28.5 g to 37.2 g after five run cycles, and the biomass content was calculated as ca. 8.7 g. So, as a modifier in the TiO₂/SA system, associated with SA, the TiO₂ was responsible for the mass/energy transfer, the higher mechanical strength and stability of the beads, resulting in more active biomass and a higher treatment efficiency, which was in good agreement with the results reported by de-Bashan and Bashan that the increasing biomass was responsible for the increasing treatment efficiency.⁴⁵

3.5 Dye-sensitized promotion mechanisms in the TiO₂/SA system

As a classical semiconductor catalyst, illumination of TiO₂ with light shorter than 400 nm can generate excess electrons in the conduction band (e_cb⁻) and positive “holes” in the valence band (h_vb⁺). The following photo-assisted reactions could occur:^47,48

TiO₂ + hν → e_cb⁻ + h_vb⁺ (1)

This is followed by the formation of extremely reactive radicals (such as HO˙) at the semi-conductor surface and/or direct oxidation of contaminants (R):

h_vb⁺ + H₂O(ads.) → HO˙ + H⁺ (2)

h_vb⁺ + OH⁻(sur.) → HO˙ (3)

h_vb⁺ + R(ads.) → R⁺ (4)

The electrons and holes may also recombine together without electron donors or acceptors:

e_cb⁻ + h_vb⁺ → TiO₂ (5)

It has been found that in an aqueous TiO₂ dispersion under irradiation by visible light, the dyes can be easily decomposed photochemically by visible light.^49,50 Photosensitized degradation of organic dyes has been carried out on TiO₂ where the organic dye serves as both a sensitizer and a substrate to be degraded.^51–57

Thus, in the TiO₂/SA system, the following reaction mechanisms are further proposed due to the dye sensitization effect (eqn (6)–(8)). That is, the electron from the excited dye molecule is injected into the conduction band of the TiO₂, and the cation radical formed at the surface quickly undergoes degradation to yield stable products:^53–55

Methyl orange/TiO₂ + visible light → methyl orange*/TiO₂ (6)

Methyl orange*/TiO₂ → methyl orange˙⁺/TiO₂ + e_cb⁻ (7)

Methyl orange˙⁺ → products (8)

HO˙ scavenging studies were conducted to determine whether the dye sensitized process was mediated by HO˙. Fig. 11a indicates that the decolorization and COD removal efficiency (100% and 52.7% respectively) in the TiO₂/SA system, were decreased by 17.0% and 13.0% after the addition of 2-propanol. The results indicate that HO˙ might be one of the active species in the TiO₂/SA system. Furthermore, Ndjou’ou et al.⁴³ and Howsawkeng et al.⁵⁸ have reported that it is possible for bacteria to coexist with HO˙ to promote simultaneous chemical and biological oxidation. Therefore, it is reasonable to deduce that the TiO₂ assisted photo-catalytic process potentially cooperated with the microbial process that contributed to the removal of methyl orange simultaneously.

Fig. 11 A comparison of the treatment efficiency in the TiO₂/SA system, the TiO₂/SA system that was scavenged by 2-propanol and the SA system after 96 h degradation (a); IR spectra of original SA, TiO₂ and TiO₂/SA (b).

Fig. 11a further demonstrated that the treatment efficiencies (83.0% decolorization and 39.7% COD removal efficiency) in the TiO₂/SA system with the addition of 2-propanol were still 6.5% and 4.1% higher than those of the SA system (76.5% and 35.6%), and it could be deduced that other promotion mechanisms should exist.

3.6 Complex-mediated photocatalysis mechanisms in the TiO₂/SA system

Complexation of ligand-to-metal charge transfer (LMCT), in which sensitization, where an electron is photo-excited directly from the highest occupied molecular orbital (HOMO) level of the adsorbate to the TiO₂ e_cb, is an easy way to extend the light response of TiO₂ to the visible region and is versatile because numerous adsorbates are potential candidates for the LMCT sensitization. A variety of organic or inorganic compounds that can form LMCT complexes on TiO₂ have been recently reviewed.^53,59

It has been reported that TiO₂–glucose could form a LMCT complex and that absorbed visible light has been recognized.⁶⁰ In the work, SA is a natural macromolecular polysaccharide (C₆H₇0₆Na)_n, which includes an abundance of –OH.^57,61 So it is reasonable to deduce that in the TiO₂/SA system, TiO₂ and SA could form a kind of complexation of ligand-to-metal (TiO₂–SA), and SA could serve as an electron donor for the reduction of N=N, contributing to the decolorization and degradation of methyl orange.

To confirm the mechanisms of LMCT which occurred in these cases, the IR spectra of the initial TiO₂, SA and the mixture of TiO₂–SA were comparatively investigated. As shown in Fig. 11b, the main bands of SA were located at 3396 cm⁻¹, 2936 cm⁻¹, 1646 cm⁻¹ and 1427 cm⁻¹, 1147 cm⁻¹ and 1015 cm⁻¹, which was assigned to the –OH stretch vibration, the C–H stretch vibration; the –COO special vibration; the C–O and C–H vibration of the pyranoid ring; the C–OH stretch vibration, respectively. The main bands of TiO₂ at 3422 cm⁻¹, 1631 cm⁻¹ and 1079 cm⁻¹ showed that the surface of the TiO₂ was loaded with –OH. Whereas, the mixture of TiO₂ and SA contributed to a series of new IR peaks compared with that of TiO₂ or SA alone, and the bands at 3000–3700 cm⁻¹ and 1000–1225 cm⁻¹ were greatly broader than those of SA and TiO₂ alone. This result could be attributed to the –OH of TiO₂ associated with the –OH, –COO and C–O of SA, which averaged out the electron cloud density, forming a LMCT complex.^40,53 Moreover, it was found that the IR spectra of TiO₂/SA were generally red shifted. The results further supported that LMCT complexation mechanisms are an easy way to extend the light response of TiO₂ to the visible region,^40,59,60 and thus, the TiO₂/SA immobilization system was much more feasible for light harvesting for cell growth than that of the SA immobilized system. Therefore, in the TiO₂/SA immobilized system, TiO₂ acted as the immobilization support and photocatalyst as well. Moreover, TiO₂ played a great role in enhancing the stability of sodium alginate.^31,38 The TiO₂–SA surface complex could be deduced in the following way.^53,59,60

Ti(IV)–OH + HO–C₆H₅O₄Na–OH → Ti(IV)–O–C₆H₅O₄Na–OH + H₂O (9)

Besides the above mechanisms, TiO₂ and SA potentially served as an adsorbent to facilitate the transport of methyl orange and energy towards both the surface and interior regions of the immobilized beads.^31,38 Thus, it was easy to explain why TiO₂, SA and TiO₂/SA alone in the dark still showed treatment efficiency for methyl orange.

4. Conclusions

In summary, a TiO₂/SA process was considered to be a novel and effective strategy for methyl orange removal. The results revealed that the TiO₂ played a pivot role in enhancing the characteristics of SA:

(1) Promoting the biomass production due to the formation of a TiO₂–SA complex that is feasible for light harvesting.

(2) Enhancing the degradation ability and stability of the immobilized beads due to the high efficiency of decolorization and COD removal in the continuous run cycles.

(3) Constructing synergetic mechanisms for the degradation of methyl orange including adsorption, biodegradation, dye sensitization and LMCT.

Additionally, the limited COD reduction could be overcome by combining the TiO₂/SA and activated sludge process.

Acknowledgements

This work was supported by the National Natural Science Foundation of China (Grant no. 51078128) and Shandong Natural Science Foundation (Grant no. Y2008B14).

Notes and references

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Footnote

† Tingting Shen and Chengcheng Jiang authors contributed equally to the work.

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